The overall objective is to gain a more complete understanding of the structure of apolipoprotein (apo) E, especially as it relates to the ability of the protein to bind to the low density lipoprotein (LDL) receptor and lipids. Point mutations are known to give isoforms of apo E that function abnormally in cholesterol and triglyceride transport. A range of physical-biochemical techniques will be used with apo E engineered and expressed in E. coli to address 3 specific aims. 1) To understand how interaction with phospholipid (PL) changes the conformation of apo E3 so that it can bind to the LDL receptor. The microenvironments of the lysine (K) residues in lipoprotein-associated, amphipathic, a-helices reflect interactions between helices so the pKa of each 13C-labeled K residue in PL-protein discoidal particles will be determined by (1H, 13C)-heteronuclear multiple quantum coherence NMR. The hypothesis to be tested is that the a-helix lengths and spacings induced by interaction with the lipid are critical for achieving high affinity binding to the LDL receptor. 2) To understand the mechanisms responsible for the differing affinities of apo E isoforms for variously-sized serum lipoprotein particles, the thermodynamic parameters characterizing the binding of apo E and engineered variants to a range of lipid particles of defined sizes will be measured using fluorescence spectroscopy and titration calorimetry. 3) To understand how the microrenvironment in the LDL receptor- binding domain of the apo E molecule correlates with the energetics of binding, the microenvironments of K143 and K146 which are directly involved in receptor binding will be investigated using apo E in states where its free energy of binding to the receptor varies widely. Changes in the receptor binding domain will be induced by either mutagenesis of the apo E molecule or changes in the associated lipid. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apo E structure and polymorphism affect the functional properties of the protein in both physiological and pathological conditions.